GSM7118671: Cre-P61-d112-Live; Homo sapiens; RNA-Seq
Organoids were rinsed 3 times with HBSS (10X HBS salt, 1M HEPES, 0.004M NaHCO3 diluted to 1X), then minced into small pieces and transferred to a 15mL conical tube for incubation in digestion solution (consisting of HBSS, 1 mg/mL Papain, 0.5mM EDTA, and 1mM L-cysteine) for 15 minutes at 37°C. Upon incubation, digestion mixture containing organoids were gently triturated with DNase I (25 μg/mL, Worthington-Biochem) and subsequently incubated again for another 10 minutes at 37°C followed by filtration with 70uM and 30uM filters (Miltenyi Biotech). Cell mixture was then centrifuged and pelleted. Cell pellet was resuspended in Neurobasal media with B27. This step was done to help dilute any remaining enzyme, EDTA, and other components of the digest mix. After the final re-suspension in Neurobasal media (without supplements), single cell mix was filtered again using 40 μM FlowMi pipet (Milipore Sigma) to help remove debris. Following isolation of single cells from brain organoids, cells were centrifuged at 300 g for 5 min and then re-suspended in 1 mL ice-cold Neurobasal media. Cell concentration and viability were determined using a hemocytometer with trypan blue dye exclusion and cell concentrations were adjusted to 700-1200 cells/μL for 10X single cell sequencing. For each sample, 9,600 cells were loaded into the 10X Chromium controller to target recovery of 6,000 cells and a Gel Beads in Emulsion (GEM) was generated. 10X Genomics 3'v3.1 chemistry was used. The samples were processed according to the protocol from 10X Genomics, using 14 cycles for cDNA amplification. Single cell libraries were sequenced using the Illumina NovaSeq 6000.